Molecular Mechanisms of Major histocompatibility complex (MHC) gene regulation

主要组织相容性复合体 (MHC) 基因调控的分子机制

• 批准号: RGPIN-2016-05455
• 负责人: Steimle, Viktor
• 金额: $ 2.26万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=615704
• 关键词: Molecular; Mechanisms; Major; histocompatibility; complex

My research program is focused on the multiple regulatory mechanisms that control major histocompatibility complex (MHC; HLA in human) class I (MHC-I) and class II (MHC-II) gene and protein expression. MHC molecules are antigen-presenting molecules displaying peptide antigens to T cells, and are thus of central importance for the adaptive immune response. MHC-dependent immune responses are intricately regulated. The MHC-II transactivator CIITA, a member of the NLR (nucleotide binding and leucine rich repeats) family of proteins, is the master regulator of MHC-II gene expression. We have found recently that protein turnover of CIITA is directly linked to its capacity to activate transcription. The first ten amino acids of CIITA isoform III act as a portable degron and transactivation sequence. The N-terminal end of CIITA isoform III is responsible for increased interaction with components of the transcription machinery. These experiments reveal a novel function of free N-terminal ends of proteins in degradation-dependent transcriptional activation. We have recently shown that NLRC5 is important for MHC-I expression. Intriguingly, NLRC5 is also an NLR protein, a family of proteins that are predominantly involved in innate immune responses. Thus far, CIITA and NLRC5 are the only NLR proteins that are transcriptional regulators. Using domain-swap experiments, we have shown that the NLRC5 N-terminal domain (NTD) behaves as a bona fide transcriptional activation domain, although its mechanism of transactivation is unknown. In collaboration with the group of Dr. T. Kufer, we propose to use our domain-swap constructs to analyze chromatin modifications at endogenous MHC-I and MHC-II promoters via ChIP (chromatin IP) experiments. We will use the NLRC5 NTD as a substrate to identify binding partners by affinity purification and mass spectrometry. Recently we have found that there is a crosstalk between IFN-γ induced HLA-II activation and estrogen receptor (ER) signaling. We propose to analyze the molecular mechanisms of this crosstalk. Bio-informatics analysis of ChIp-seq data revealed in vivo binding of ERa at several distal sites around the CIITA gene, which in part overlap with distal regulatory elements of IFN-γ induced CIITA expression. At a number of sites, several potentially relevant transcription factors, such as Era, FoxA, Stat1, IRF1 and BRG1 bind in close proximity. We will carry out a ChIp-seq and RNA-seq analysis for ERa, FoxA1 and IRF1 in breast cancer cells treated simultaneously with IFN-γ and E2. Functional tests of potential regulators will be carried out by shRNA-mediated knockdowns. Potential regulatory sites will be analyzed by somatic knockouts of potential endogenous distal regulatory sites. Taken together these experiments will inform us on the interactions of important signaling pathways in general and on the regulation of the CIITA gene in particular.

我的研究重点是控制主要组织相容性复合体(MHC；人类的人类白细胞抗原)I类(MHC-I)和II类(MHC-II)基因和蛋白表达的多种调控机制。MHC分子是抗原递呈分子，向T细胞展示多肽抗原，因此对获得性免疫反应至关重要。依赖MHC的免疫反应受到错综复杂的调控。MHC-II反式激活因子CIITA是NLR(核苷酸结合和富含亮氨酸重复序列)家族的成员，是MHC-II基因表达的主要调节因子。我们最近发现，CIITA的蛋白质周转与其激活转录的能力直接相关。CIITA亚型III的前10个氨基酸是一个可携带的降解和反式激活序列。CIITA亚型III的N端负责增加与转录机器组件的相互作用。这些实验揭示了蛋白质的游离N末端在降解依赖的转录激活中的一种新功能。 我们最近发现NLRC5对MHC-I的表达很重要。有趣的是，NLRC5也是一种NLR蛋白，这是一个主要参与先天性免疫反应的蛋白质家族。到目前为止，CIITA和NLRC5是仅有的NLR转录调节蛋白。通过结构域交换实验，我们证明了NLRC5的N-末端结构域(NTD)是一个真正的转录激活结构域，尽管它的转录激活机制尚不清楚。在与T.Kufer博士的团队合作下，我们建议通过CHIP(染色质IP)实验，使用我们的结构域交换结构来分析内源MHC-I和MHC-II启动子上的染色质修饰。我们将以NLRC5 NTD为底物，通过亲和纯化和质谱法鉴定结合伙伴。 最近，我们发现干扰素-γ诱导的人类白细胞抗原-II激活与雌激素受体(ER)信号之间存在串扰。我们建议分析这种串扰的分子机制。ChIP-SEQ数据的生物信息学分析表明，ERA在体内结合在CIITA基因周围的几个远端位置，部分与干扰素-γ诱导的CIITA表达的远端调控元件重叠。在许多位点，几个潜在的相关转录因子，如ERA、FoxA、STAT1、IRF1和BRG1紧密结合。我们将对同时用干扰素-γ和E2处理的乳腺癌细胞进行Era、FoxA1和IRF1的芯片序列和RNA-SEQ分析。潜在调控因子的功能测试将通过shRNA介导的击倒来进行。潜在的调控位点将通过对潜在的内源性末端调控位点的体细胞敲除来分析。总的来说，这些实验将使我们了解重要信号通路的相互作用，特别是对CIITA基因的调控。